胃癌干细胞单克隆抗体联合顺铂靶向治疗胃癌的实验研究

目的:探讨靶向胃癌干细胞单克隆抗体的生物学特征及其联合顺铂治疗胃癌的疗效.方法:采用无血清悬浮培养法和PKH26染色法确定胃癌SNU-5细胞中存在肿瘤干细胞.细胞免疫荧光法检测单克隆抗体21A3识别的抗原蛋白与PKH26+细胞及特异性抗原(CD90)在SNU-5细胞中的表达情况.无血清悬浮培养法检测流式细胞术分选出的21A3+细胞的自我更新能力以及21A3对SNU-5细胞自我更新能力的影响.CCK8法检测21A3对细胞顺铂耐药能力的影响.裸鼠体内治疗实验分析21A3联合顺铂对SNU-5移植瘤生长的抑制作用.结果:SNU-5细胞无血清悬浮培养11d后形成的细胞球体中存在单个PKH26+细胞.细胞免疫荧光显示,单克隆抗体21A3识别的抗原分子能与PKH26和CD90在SNU-5细胞上共定位.21A3+细胞体外成球率高于21A3-细胞.21A3+细胞具有更高的耐药性,21A3+细胞和21A3-细胞的IC 50分别为0.104μmol/L和0.025μmol/L.单克隆抗体21A3能够显著抑制SNU-5细胞的无血清成球,抑制率为37.5%.经21A3处理后的SNU-5细胞,耐药能力下降,其IC 50为0.001μg/ml,而对照组的IC 50为0.003μg/ml.抗体体内治疗实验结果显示,10、5、2.5mg/kg的21 A3组的肿瘤生长抑制率分别为80.6%、69.6%和59.2%,10mg/kg 21A3+顺铂组的肿瘤生长抑制率为90.6%,顺铂组的肿瘤生长抑制率为55.8%.结论:单克隆抗体21A3是抗胃癌干细胞的功能性单克隆抗体.

Experimental study on the treatment of gastric cancer by targeting specific antigen posi-tive gastric cancer stem cell monoclonal antibody in combination with cisplatin

Objective:To investigate the biological characteristics of monoclonal antibodies against human gastric cancer stem cells and the therapeutic effect of combined with cisplatin in the treatment of gastric cancer. Methods:Cell culture in serum - free medium and PKH26 staining were used to determine the existence of cancer stem cell in SNU - 5 cell line. The co - expression of antigen recognized by McAb 21A3 and CD90 or PKH26 positive cells were detected by immunofluorescence assay. Serum - free suspension culture was used to detect the self - renewal ability of SNU - 5 and 21A3 positive cells sorted by flow cytometry and SNU - 5 treated by 21A3. The effect of 21A3 on cispla-tin resistance was examined by CCK8. The inhibition of implanted tumor growth and spontaneous lung metastasis of 21A3 combined with cisplatin were studied by tumor treatment experiments and the survival of mice was also ob-served. Results:The single PKH26 positive cell was observed in spheroids when SNU - 5 cells had been cultured for 11 days. The immunofluorescence assay showed that 21A3 could recognize cells which were stained by PKH26 and FITC - conjugated CD90 antibody. The self - renewal capability of 21A3 positive cells in serum - free medium was significantly higher than 21A3 negative cells. The cisplatin resistance of 21A3 positive cells was obviously higher than 21A3 negative cells. In vitro functional experiments demonstrated that McAb 21A3 significantly suppressed the sphere formation of SNU - 5 cells,with the inhibitory rate being 36. 4% . 21A3 also notably inhibited the cisplatin resistance of SNU - 5 cells. The IC50 was 0. 001μg/ ml in 21A3 group,and 0. 003μg/ ml in control group. Animal experiment re-vealed that 10mg/ kg,5mg/ kg,2. 5mg/ kg - dose of 21A3 and can significantly inhibit the tumor growth,and the inhib-itory rate was 80. 6%,69. 6% and 59. 2%,respectively. Furthermore,the inhibitory rate of high - dose McAb 21A3 combining with cisplatin and cisplatin monotherapy were 90. 6% and 55. 8% . Conclusion:McAb 21A3 is one of functional monoclonal antibodies targeting gastric cancer stem cells.